Systems and methods for processing cells

ABSTRACT

The present invention efficiently and cost-effectively extracts and collects cells from a tissue. The inventors have discovered that the tissue can be effectively fragmented and the resulting cells can be purified using a system or kit with multiple components. An advantage of the present invention is that tissue processing takes place in a closed system such that sterility can be maintained throughout the process, even if certain components are removed during processing, for example through the use of valves, clamps, and heat seals. Furthermore, any or all of the steps can be automated or manually accomplished, according to the specific needs of the application or the user.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of, claims priority to and thebenefit of, and incorporates herein by reference in its entirety U.S.patent application Ser. No. 14/547,966, which was filed on Nov. 19, 2014and which is a divisional of U.S. patent application Ser. No.13/933,034, which itself was filed on Jul. 1, 2013 and issued on Nov.25, 2014 as U.S. Pat. No. 8,893,995. U.S. patent application Ser. No.13/933,034 is a continuation of International Patent Application No.PCT/US2012/064130, which was filed on Nov. 8, 2012 and which claimspriority to and the benefit of U.S. Provisional Patent Application No.61/557,127, which itself was filed on Nov. 8, 2011. The contents of U.S.patent application Ser. No. 13/933,034, International Patent ApplicationNo. PCT/US2012/064130, and U.S. Provisional Patent Application No.61/557,127 are also incorporated herein by reference in their entirety.

TECHNICAL FIELD

In various embodiments, the present invention relates to systems andmethods for processing tissue to isolate and collect target cells.

BACKGROUND OF THE INVENTION

Purification of viable cells from a tissue sample can be a laboriousprocess that involves dissection and other manual manipulation andprocessing steps, as well as, in some cases, cell culturing. Maintainingsterility of the cells during the purification process is also animportant concern. Although laminar hoods can be used to maintainsterility, they suffer a number of disadvantages. For example, suchhoods are expensive, relatively immobile, cumbersome to work with, andconsume valuable laboratory space. Efficiency of the cell purificationprocess is another concern that further complicates the purificationprocess. Isolating rare cells, such as stem cells, from a tissue samplerequires an efficient process to recover as many of the cells aspossible.

There remains a need for a practical, cost-effective, sterile, andefficient mechanism and method for extracting and collecting cells, suchas stem cells, for advancing potential therapies that rely on theadministration of these cells.

SUMMARY OF THE INVENTION

The present invention efficiently and cost-effectively extracts andcollects cells from a tissue. The inventors have discovered that thetissue can be effectively fragmented and the resulting cells can bepurified using a system or kit with multiple components. An advantage ofthe present invention is that tissue processing takes place in a closedsystem such that sterility can be maintained throughout the process,even if certain components are removed during processing, for examplethrough the use of valves, clamps, and heat seals. Furthermore, any orall of the steps can be automated or manually accomplished, according tothe specific needs of the application or the user.

Thus, in one aspect, the invention relates to a tissue mincing tool. Thetissue mincing tool includes a compartment for a tissue sample, acutting surface at one end of the compartment, and a sterile, sealedcontainer. The cutting surface separates the compartment from thesterile, sealed container, such that a tissue sample that passes throughthe cutting surface can be deposited within the container. The cuttingsurface can be dimensioned to mince the tissue sample into fragmentshaving an average cross-section no greater than four square millimeters.For example, the number of square millimeters may be no greater than 3,2, 1, 0.5, 0.3, 0.2, 0.1, or 0.05 in various embodiments of theinvention. The tissue mincing tool can also include a second cuttingsurface for further reducing the average cross-section of the fragments.The cutting surface of the tissue mincing tool can include an automatedcutting system. For example, the cutting surface may includesemi-automatic scissors. One or more mincer screens may be positioned inproximity to the cutting surface. The tissue mincing tool may furtherinclude a suction cup for stabilizing the tool during operation thereof.The tissue mincing tool also or alternatively can include a fluidconduit in communication with the sterile, sealed container, and aseparator unit such as one or more filters within the fluid conduit. Thesterile, sealed container optionally includes at least one sealed accessport permitting the sterile introduction of a fluid into the container.

In the tissue mincing tool, the compartment for the tissue sample canincorporate one or more features to facilitate the application of aforce to the tissue sample, impelling it beyond the cutting surface andinto the sterile, sealed container. For example, a portion of thecompartment can be shaped to receive a solid member to press the tissuesample. Such a solid member can optionally be included with the tissuemincing tool, whether connected to it or provided as a separatecomponent.

In one embodiment, a portion of the compartment near the cutting surfacehas a substantially constant cross-section, such that a comparablyshaped solid member may be introduced into the compartment and fill thatportion, while pressing the tissue sample into or through the cuttingsurface. In another embodiment, the compartment near the cutting surfacehas a tapered or cone-shaped end. In some embodiments, an interiorsurface of the compartment is threaded, such that a threaded solidmember may be guided into the compartment. In some embodiments, thecompartment also includes a gasket, which can provide an improved sealwhen a solid member is introduced into the compartment. In yet anotherembodiment, the tissue mincing tool includes a shaft crank for movingthe cutting surface towards the tissue sample.

The invention also provides methods of using any of the above-describedtissue mincing tools by impelling the tissue sample through the cuttingsurface of the tool. The invention provides methods of mincing a tissuesample and optionally injecting an enzyme into the sterile, sealedcontainer, such that the enzyme enhances the digestion of the mincedtissue. The enzyme can be a protease, such as collagenase,hyaluronidase, or dispase, separately or in combination. These steps canoptionally be incorporated into a method of separating cells from thetissue sample by mincing and/or digesting the tissue sample and removingfragments larger than about 40 microns (e.g. fragments retained by afilter having a pore size of about 500 microns, or fragments retained bya filter having a pore size of about 300 microns, or fragments retainedby a filter having a pore size of about 250 microns, or fragmentsretained by a filter having a pore size of about 150 microns, orfragments retained by a filter having a pore size of about 100 microns,or fragments retained by a filter having a pore size of about 70microns, or fragments retained by a filter having a pore size of about40 microns). These larger fragments, referred to herein as “undigestedtissue,” can be removed by filtering or sedimentation. These methods areeffective for purifying cells from any of a variety of solid tissues.For example, the methods described herein can separate cells, such asstem cells, from fat tissue or afterbirth tissue, such as placental orumbilical cord tissue or, more specifically, a tissue comprisingWharton's Jelly. In some embodiments, the tissue sample is substantiallyfree of blood vessels, which can optionally be dissected from a tissuebefore the tissue is placed in the compartment.

In another aspect, the invention relates to a cell collection methodincluding sedimenting cells in a cell collection device. The cellcollection device includes a sterile container for a fluid includingcells and a fluid passageway in communication with the sterilecontainer. The fluid passageway includes a cell capture zone, such thatthe volume of the cell capture zone can be less than 5% of the volume ofthe sterile container and the sterile container and the cell capturezone can be configured such that sedimentation of the cells from thefluid in the sterile container concentrates the cells in the cellcapture zone. This aspect or any of the following aspects can have anyof the following embodiments. The cell collection device furtherincludes a second sterile container in communication with the fluidpassageway and/or a removable clamp to regulate passage of material intothe second sterile container. The second sterile container can beheat-sealable and/or a bag. The cell collection method can also includecentrifuging the cell collection device to accelerate sedimentation ofthe cells, and the cells can be Wharton's Jelly stem cells. The fluidincludes mechanically minced and/or enzymatically digested umbilicalcord tissue. The cell collection method can further include adding acryoprotectant to the cells; the cryoprotectant includes DMSO, albumin,and/or dextran. The method can also include adding autologous plasma tothe cells.

In still another aspect, the invention relates to a cell collectiondevice having a fluid including cells, a sterile container for housingthe cells, and a fluid passageway in communication with the sterilecontainer. The fluid passageway includes a cell capture zone, whereinthe volume of the cell capture zone can be less than 5% of the volume ofthe sterile container and the sterile container and the cell capturezone can be configured such that sedimentation of the cells from thefluid in the sterile container concentrates the cells in the cellcapture zone. This aspect or any of the following aspects can have anyof the following embodiments. The cell collection device also oralternatively includes a second sterile container in communication withthe fluid passageway and/or a removable clamp to regulate passage ofmaterial into the second sterile container. The second sterile containercan be heat-sealable and/or a bag.

In yet another aspect, the invention relates to a cell collection devicehaving a sterile container for a fluid comprising cells, such that thesterile container can be adapted for use in sedimentation, and a fluidpassageway in communication with the sterile container. The fluidpassageway includes first and second valves defining a cell capture zonetherebetween, such that the volume of the cell capture zone can be lessthan 5% of the volume of the sterile container and the sterile containerand the cell capture zone can be configured such that sedimentation ofthe cells from the fluid in the sterile container concentrates the cellsin the cell capture zone. This aspect or any of the following aspectscan have any of the following embodiments. The sterile container can beadapted for use in centrifugation. The cell collection device also oralternatively includes a second sterile container in communication withthe second valve and/or a removable clamp to regulate passage ofmaterial into the second sterile container. The second sterile containercan be heat-sealable and/or a bag.

In still yet another aspect, the invention relates to a cell collectiondevice having a sterile container for housing the cells, a fluidpassageway in communication with the sterile container, and a secondsterile container in communication with the fluid passageway. The fluidpassageway includes a cell capture zone, such that the volume of thecell capture zone is less than 5% of the volume of the sterile containerand the sterile container and the cell capture zone can be configuredsuch that sedimentation of the cells from the fluid in the sterilecontainer concentrates the cells in the cell capture zone. The secondsterile container includes an element selected from the group consistingof a bag, a heat-sealable container, and a removable clamp.

In another aspect, the invention relates to cell collection device thatincludes a compartment for receiving a tissue; a cutting surfacedimensioned to mince the tissue sample into fragments having an averagecross-section no greater than four square millimeters; a sterile, sealedcontainer for holding a suspension of the minced tissue, the sterilecontainer having a volume at least ten times greater than the volume ofthe compartment for receiving a tissue; a filter bag in fluidcommunication with the sterile, sealed container; and a sedimentationbag in fluid communication with the filter bag. The filter bag containsat least one filter having a pore size sufficiently small to retainparticles larger than about 250 μm. The sedimentation bag includes atapered portion to promote the concentration of sedimented cells.

BRIEF DESCRIPTION OF THE FIGURES

Other features and advantages of the present invention, as well as theinvention itself, can be more fully understood from the followingdescription of the various embodiments, when read together with theaccompanying drawings, in which:

FIG. 1 schematically depicts a tissue mincing tool, according to anembodiment of the invention;

FIG. 2 is a schematic perspective view of a solid member of a tissuemincing tool, according to an embodiment of the invention;

FIG. 3 is a schematic perspective view of the solid member of FIG. 2;

FIG. 4. is a schematic perspective view partially showing a cuttingsurface, according to an embodiment of the invention;

FIGS. 5A and 5B are schematic views partially showing a solid member andits complementary compartment, according to embodiments of theinvention;

FIG. 6A is a side view of a tissue mincing tool, according to anotherembodiment of the invention;

FIG. 6B is a cross-sectional view along the line E-E of the tissuemincing tool of FIG. 6A;

FIG. 6C is an exploded view of the tissue mincing tool of FIG. 6A;

FIG. 7 is a schematic perspective view of a tissue mincing tool,according to yet another embodiment of the invention;

FIG. 8 provides an overview of the procedural steps for collecting andisolating desired cells from a tissue sample, according to an embodimentof the invention;

FIG. 9 provides an overview of the procedural steps for collecting andisolating desired cells from a tissue sample, according to an embodimentof the invention; and

FIGS. 10A-10G depict procedures for collecting and isolating desiredcells from a tissue sample.

DETAILED DESCRIPTION

To provide an overall understanding of the invention, certainillustrative embodiments will now be described, including systems andmethods for processing cells. However, it will be understood by one ofordinary skill in the art that the systems and methods described hereinmay be adapted and modified as is appropriate for the application beingaddressed and that the systems and methods described herein may beemployed in other suitable applications. All such adaptations andmodifications are to be considered within the scope of the invention.Furthermore, it is to be understood that the features of the variousembodiments described herein are not mutually exclusive and can exist invarious combinations and permutations.

Tissue Mincing Tool

FIG. 1 schematically depicts a tissue mincing tool 10 according to oneembodiment of the invention. The tissue mincing tool 10 includes acompartment 12 within which a tissue sample can be received and beinitially housed, and a sterile, sealed container 14. The compartment 12extends between first and second ends 16, 18, while the sterile, sealedcontainer 14 itself extends between first and second ends 18, 20. Acutting surface 108 is located at the second end 18 of the compartment12 or, equivalently, at the first end 18 of the sterile, sealedcontainer 14, and therefore separates the compartment 12 from thesterile, sealed container 14. As illustrated, the tissue mincing tool 10also includes a solid member 100. The solid member 100 is depicted to bepartially located within (as shown in phantom) the compartment 12. Apart of the solid member 100 is also shown to be located outside thecompartment 12. Threads 105 are depicted on that exterior part of thesolid member 100.

FIG. 2 depicts an embodiment of the solid member 100 of the tissuemincing tool 10. Other portions of the tissue mincing tool 10 (e.g., thecompartment 12 and the sterile, sealed container 14) are not shown. Thesolid member 100 includes a larger diameter plunger 104 (shown with thethreads 105) and a smaller diameter screw shaft 106. A handle 114 isdisposed at one end of the solid member 100. At the opposite end of thesolid member 100, the cutting surface 108 is attached to the end of thesmaller diameter screw shaft 106. As illustrated, the smaller diameterscrew shaft 106 extends from the cutting surface 108 to the handle 114(to which the smaller diameter screw shaft 106 is also coupled) and, inso doing, the smaller diameter screw shaft 106 runs through a hollow(e.g., central) portion 107 of the larger diameter plunger 104. In oneembodiment, the screw shaft 106 features threads 109 along a certainlength thereof and the plunger 104 includes complementary grooves 111within its hollow portion 107. In this way, the plunger 104 isthreadably engaged with the screw shaft 106. Accordingly, when thehandle 114 is rotated, the screw shaft 106 is also caused to rotate, asis (optionally) the cutting surface 108 that is attached to the end ofthe screw shaft 106. Moreover, rotation of the screw shaft 106 causestranslation of the plunger 104, for example towards the cutting surface108. A tissue sample housed within the compartment 12 between theplunger 104 and the cutting surface 108 may thus be pressed into contactwith the (optionally rotating) cutting surface 108 and, as describedfurther below, cut thereby.

The threaded portion 105 of the larger diameter plunger 104 can engageand mate with a complementary threaded interior portion of thecompartment 12 that receives the tissue sample. Threaded engagementbetween the plunger 104 and the compartment 12 provides for leverage andcontrol so that a user can more easily translate the plunger 104 withinthe compartment 12 using the handle 114, as well as slide or push thetissue sample towards and through the cutting surface 108. Asillustrated in FIG. 1, the plunger 104 is sized to mate with andsubstantially fill an interior hollow cavity of the compartment 12. Inan embodiment, with reference again to FIG. 2, a gasket 118 may bedisposed at one end of the plunger 104. Together, the gasket 118 andplunger 104 may substantially fill an inner diameter of the interiorhollow cavity of the compartment 12, thereby creating an air-tight,vacuum seal in the compartment 12. Gasket 118 may be substantiallycomplementary in shape to cutting surface 108. For example, the bottomsurface of the gasket and the top surface of the cutting surface 108 canboth be round or flat. In an embodiment, gasket 118 forms a flatsurface. A tissue sample is impelled through cutting surface 108 and,thus, minced when gasket 118 contacts and drives the tissue sample.

The cutting surface 108 can be any surface configured to cut, parse, orseparate a tissue sample into smaller portions without damaging thecells from the tissue sample when the tissue sample is pushed throughthe cutting surface 108. For example, the cutting surface 108 can mincea tissue sample into smaller portions with an average cross-section nogreater than four square millimeters, or one square millimeter, thoughcutting surfaces that can mince the tissue sample into larger or smallerportions are contemplated. A tissue mincing tool can include a secondcutting surface to further reduce the average cross-section of theminced tissue samples. Examples of a cutting surface include a gratewith sharp edges, multiple sharp wires across an opening, a steel plateor disc with multiple holes resting on a lip inside a compartment, holesin a plate that are offset and have a sharp edge, and a mesh of sharpsurfaces defining apertures. Additionally or alternatively, in anembodiment in which a cutting surface defines apertures, an end of theplunger 104 can form multiple projections, such as fingers, that matewith the apertures of the cutting surface 108 to assist in pushing thetissue sample through the cutting surface 108. In an alternativeembodiment, the end of the plunger 104 can be flat. Moreover, thecutting surface 108 can be textured or can form multiple projections(e.g., a cleat) to create a frictional surface or to keep the tissuesample on the cutting surface 108 as pressure is applied to the tissuesample. In a further embodiment, the cutting surface 108 may include anautomated cutting system, such as a semi-automatic scissors.

In an embodiment, an optional nose nut (not shown) may be disposed aboutthe cutting surface 108 to retain the cutting surface 108 in position asa tissue sample is being impelled therethrough. For example, theoptional nose nut can be removably attached to the compartment 12 thatreceives the tissue sample. The optional nose nut can include aprojection that engages with a recessed surface of the compartment 12 toform a snap-fit connection. Additionally or alternatively, a threadedportion of the optional nose nut can engage a similarly andcomplementary threaded portion of the compartment 12. When the optionalnose nut is fully engaged with the compartment 12, the cutting surface108 is disposed on and retained in position by a lip of the optionalnose nut.

FIG. 3 depicts a perspective view of the solid member 100 of the tissuemincing tool 10. Other portions of the tissue mincing tool (e.g., thecompartment 12 and the sterile, sealed container 14) are not shown. Asbefore, the solid member 100 includes a larger diameter plunger 104(shown with the threads 105) and a smaller diameter screw shaft 106. Thehandle 114 is disposed at one end of the solid member 100. At theopposite end of the solid member 100, the cutting surface 108 isattached to the end of the smaller diameter screw shaft 106. In oneembodiment, as illustrated, the cutting surface 108 includes a cuttingblade 110 adjacent a mincing disc 112.

FIG. 4 depicts a perspective view of an exemplary cutting surface 108 ofthe tissue mincing tool 10. The cutting surface 108 includes the cuttingblade 110. Additionally, the cutting surface 108 includes the mincingdisc 112 with apertures 116 that allow tissues samples to be impelledtherethrough.

FIGS. 5A and 5B depict two embodiments of a solid member 100 of a tissuemincing tool 10. In one embodiment depicted in FIG. 5A, the plunger 104of the solid member 100 is substantially cylindrical in shape near itsend. In a second embodiment depicted in FIG. 5B, the plunger 104 of thesolid member 100 has a tapered or cone-shaped end. In each embodiment,the solid member 100 is shaped to mate with and to fill the compartment12 that is complementarily shaped, such that the solid member 100 ismovable within the compartment 12 to impel a tissue sample disposedwithin the compartment 12 through the cutting surface 108. As the tissuesample passes through the cutting surface 108, the cut portions thereofare deposited within the sterile, sealed container 14, which may be, forexample, a bag. Additionally or alternatively, the interior cavity ofthe compartment 12 can define a recessed channel or surface (e.g., a camsurface) to engage a projection protruding from the plunger 104 suchthat solid member 100 can be removably secured to the compartment 12 andthe user still has leverage and control to translate the solid member100 into the compartment 12 (not shown).

FIG. 6A depicts a side view of a tissue mincing tool 200 according toanother embodiment of the invention. FIG. 6B depicts a cross-sectionalview along the line E-E of the tissue mincing tool 200 shown in FIG. 6A,while FIG. 6C depicts an exploded view of the tissue mincing tool 200shown in FIG. 6A. The tissue mincing tool 200 includes a base 204, areservoir 208, and a handle 212. As shown, the base 204 may include athreaded portion 216 for threadably engaging the reservoir 208. Forexample, an interior surface of the reservoir 208 may feature grooves218 that complement the threaded portion 216 of the base 204. Thus,during assembly of the tissue mincing tool 200, the reservoir 208 may bescrewed onto the base 204.

As illustrated, a suction cup 220 may be coupled to a bottom portion 224of the base 204. In one embodiment, the suction cup 220 providesstability for the tissue mincing tool 200. For example, in operation, auser may couple the tissue mincing tool 200 to a table (or other supportsurface) using the suction cup 220. Stability is thereby provided to thetissue mincing tool 200 when, for example, the user turns the handle 212as further described below, or otherwise imparts force to the tissuemincing tool 200.

As most clearly shown in FIG. 6B, included within the reservoir 208 is acompartment 228 for initially housing a tissue sample. In addition, atleast one cutting surface (which is coupled to the handle 212 through ashaft crack 232) is moveable within the reservoir 208. For example, asmost clearly shown in FIG. 6C, a first cutting surface 236, a firstmincer screen 240, a second cutting surface 244, and a second mincerscreen 248 (each of which are coupled to the handle 212 through theshaft crank 232) may be moveable within the reservoir 208 throughactuation (e.g., rotation) of the shaft crank 232. The shaft crank 232may, for example, be manually actuated by a user via the handle 212, or,alternatively, may be automatically machine-actuated via a separatedevice. As will be understood by one of ordinary skill in the art, thefirst and second cutting surfaces 236, 244 may be any of the exemplarycutting surfaces described above. In addition, the first and secondmincer screens 240, 248 may, as illustrated, include differently-sizedapertures.

In operation, as the shaft crank 232 is rotated, the first and secondcutting surfaces 236, 244 are likewise rotated and move downward withinthe reservoir 208 toward the tissue sample that is housed within thecompartment 228. The first cutting surface 236 makes contact with andcuts the tissue sample into one or more smaller portions. Those smallertissue portions are then passed through the apertures of the firstmincer screen 240, are again cut into even smaller portions by thesecond cutting surface 244, and are finally passed through the aperturesof the second mincer screen 248. The first and second cutting surfaces236, 244 are rotated and moved downwards within the reservoir 208 untilsubstantially all of the tissue sample (or at least a sufficient amountof the tissue sample for a given application) is minced and passesthrough the second screen 248. Upon passing through the second screen248, the minced tissue sample is collected and housed within a container252 of the reservoir 208. Although not depicted as such in FIGS. 6A-6C,the top portion of the reservoir 208 may in fact be capped and theinterior portion of the reservoir 208 sterilized, such that thecontainer 252 is a sterile, sealed container 252.

As will be understood by one of ordinary skill in the art, and asdescribed above, the compartment 228 of the reservoir 208 is, as shownin FIG. 6B, located below the first and second cutting surfaces 236, 244and first and second mincer screens 240, 248, while the sterile, sealedcontainer 252 of the reservoir 208 is located above the first and secondcutting surfaces 236, 244 and first and second mincer screens 240, 248.As such, in the embodiment of the tissue mincing tool 200 depicted inFIGS. 6A-6C, the sizes of the compartment 228 and of the sterile, sealedcontainer 252 vary as the first and second cutting surfaces 236, 244 arerotated downwards (or upwards).

As will also be understood by one of ordinary skill in the art, thedepiction of the tissue mincing tool 200 in FIGS. 6A-6C is non-limiting.In fact, variations, modifications, and other implementations arecontemplated. For example, fewer or more than two cutting surfaces 236,244 and/or two mincer screens 240, 248 may be employed. As anotherexample, the shaft crank 232 and handle 212 may be coupled such that thehandle 212 is rotatable within a vertical plane rather than a horizontalplane (as illustrated).

FIG. 7 depicts a tissue mincing tool 300, according to another view ofan embodiment of the invention, whose principal of operation is similarto that of the tissue mincing tool 200 depicted in FIGS. 6A-6C. Asbefore, the tissue mincing tool 300 includes a base 304, a reservoir308, and a handle 312. As shown, a top portion of the reservoir 308 iscoupled to a bottom portion thereof through the use of screws 315 andwing nuts 317. The tissue mincing tool 300 also includes a suction cup320 to provide the aforedescribed stability to the tool 300 duringoperation. The container 352 of the reservoir 308 within which theminced tissue sample is collected after passing through, for example, asecond screen 348 is also illustrated. As before, the top portion of thereservoir 308 may be capped and the interior portion of the reservoir308 sterilized, such that the container 352 is a sterile, sealedcontainer 352.

Cell Isolation and Collection Method

The invention also provides methods for efficient and sterile processingof tissue to isolate and to collect target cells. FIG. 8 provides anoverview of the procedural steps for isolating and collecting cells.Generally, a tissue sample can be initially minced using any of theabove-described tissue mincing tools by impelling the tissue samplethrough the cutting surface of the tool and into a sterile, sealedcontainer. The invention also provides optional methods of furtherdigesting the tissue sample by exposing it to a chemical or an enzyme.For example, the minced tissue may be digested by injecting an enzymeinto the container, such that the enzyme digests the minced tissue. Theenzyme can be a protease, such as collagenase, hyaluronidase, ordispase, separately or in combination. These steps are additionally oroptionally incorporated into a method of separating minced and/orenzymatically digested tissue sample from any larger fragments(“undigested tissue,” as described above), for example, decanting,aspiration, sedimentation, or preferably, filtering. In someembodiments, the minced and/or enzymatically digested tissue, which canbe viscous, is washed or diluted before a separating step. Theseparation of the target cells from the minced and/or enzymaticallydigested tissue can be accomplished by sedimentation of the cells from amixture containing the minced and/or enzymatically digested tissue.Although gravity sedimentation can be used, the sedimentation processcan be accelerated by, for example, centrifugation. Additionally, andalternatively, the target cells are moved into a sterile container to becryo-preserved for later use.

In an embodiment, methods for separating minced and/or enzymaticallydigested tissue sample from undigested tissue may include two or morefiltration steps as depicted in FIG. 9. For example, minced and/orenzymatically digested tissue sample may be subjected to multiplefiltration steps using filters of varying sizes. In an embodiment, theminced and/or enzymatically digested tissue sample is initiallysubjected to a first filtration step using a large-pore filter of, e.g.,about 500 microns, about 250 microns, about 150 microns or about 100microns, for removing coarse undigested tissue. Additionally, a secondfiltration step can be carried out to filter the eluate from the firstfiltration step using a small-pore filter of, e.g., about 70 microns orabout 40 microns, for removing additional contaminants such as collagenfibers. Because the minced and/or enzymatically digested tissue isgenerally viscous, the tissue can be washed or diluted with anappropriate sterile solution (such as a buffered salt solution) at anystage in the process. For example, after the minced and/or enzymaticallydigested tissue has been separated from the undigested tissue followingthe first filtration step, further washes can be performed to furthercleanse the minced and/or enzymatically digested tissue before thesecond filtration step. Following multiple rounds of filtration, targetcells substantially free of tissue sample can be collected bysedimentation.

FIG. 10A depicts an exemplary procedure for collecting and isolatingdesired cells from a tissue sample. The tissue sample may be initiallyplaced within a compartment, where it can be minced, parsed, orseparated into smaller portions. An advantage of mincing the tissuesample before any enzymatic digestion is that the entire surface area ofthe tissue sample on which the enzyme can act is increased. Thecompartment may be fitted and attached to one port (e.g., an aperture)of a container (e.g., digestion bag) such that a tissue sampleintroduced into the compartment can directly pass through into thecontainer. The compartment may or may not be removably attached to thecontainer.

The container defines a sterile, sealed interior space that holds theminced tissue sample and fluids. The container may include sealed portsfor introducing or dispensing materials and fluids into or from thecontainer. For example, the container may include one or more injectionports for introducing fluids and one or more withdrawal ports fordispensing or suction fluids and materials from the container. Further,in an alternative embodiment, each of the injection ports and withdrawalport can be configured such that fluids and materials can only be movedin one direction to and from the container. Moreover, the ports can bedisposed at an opposite end of the container from the compartment,though the ports can also be disposed along any portion of the perimeterof the container. In an embodiment, the ports are not removably securedto the container. Additionally or alternatively, syringes, air vents,capped air vents, or other devices that mate with a luer connection canbe attached to the ports. All ports may be swabbable so that sterilityis maintained.

Subsequently, the minced tissue can optionally be digested by, forexample exposing it to a chemical or an enzyme. In an embodiment, theminced tissue may be digested by an enzyme, for example, a protease,such as a collagenase, hyaluronidase, or dispase, separately or incombination. The enzyme may be directly introduced into the container,such that the enzyme digests the minced tissue. For example, a syringe,or any other device that can house fluids, materials, or air, can beconnected to the container (e.g., via a luer connection) and used todispense, for example, a protease into the container to digest theminced tissue sample. To enhance digestion of the minced tissue sample,the container can be inverted to circulate the enzyme about thecontainer. Depending on the rate of enzymatic breakdown of the mincedtissue sample, the container can be placed at rest and the minced tissuesample can be incubated with the enzyme at 37° C. for a period of time,for example, for about one to three hours, though more or less time iscontemplated, to digest the minced tissue sample. Additionally oralternatively, to assist in the incubation process, the container canoptionally be periodically mixed with an orbital shaker or moved througha series of rollers or other compression-type device to assist in thebreak-down of the minced tissue sample within the container. In anexample in which the tissue sample is about 10 mL, a user can injectabout 10 mL of enzyme into the container, though more or less enzyme iscontemplated. Once the minced tissue sample is digested, a digestedtissue sample of about 20-30 mL results.

Before the cells are separated from the minced and/or enzymaticallydigested tissue, any remaining fragments of undigested tissue areoptionally removed to facilitate the subsequent purification of thecells. Depending on their size, undigested tissue can be removed by, forexample, physical extraction, decanting, aspiration, sedimentation, orpreferably, filtering. Optionally, the undigested tissue that is removedmay be stored and/or used for other purposes such as a seeding sourcefor the expansion of stem cells.

FIGS. 10A-10D illustrate various embodiments in which separation isachieved by filtration. Specifically, FIGS. 10A-10D depict a fluidpassageway that connects the container holding the digested tissuesample to a filter unit which can be removably attached to thecontainer. The filter unit may use a single filter, or a plurality offilters, optionally of decreasing size. Alternatively, a filter may befitted and disposed in the container such that that the container isdivided into two sub-spaces. The filter may be symmetrically orasymmetrically placed within the container. Additionally oralternatively, the filter may be fitted within a port, for example, awithdrawal port. The size of the filter can be about 500 microns, about250 microns, about 150 microns, about 100 microns, about 70 microns,about 40 microns, or any range thereof, depending on the application.The digested tissue sample, which can be viscous, may be diluted priorto filtering so that the resultant tissue sample can more easily movethrough the filter into downstream containers or components for furtherprocessing. Examples of diluting solutions include phosphate bufferedsaline (PBS), 5% human serum albumin, saline, heta-starch, and freshplasma (e.g., autologous plasma). In an embodiment, a syringe, or anyother device that can house fluids, is used to dispense a dilutingsolution into the container via an injection port. In an example inwhich the digested tissue sample is about 20-30 mL, a user can injectabout 250 mL of a diluting solution into the container, though more orless solution is contemplated. As a result, the container holds about250-300 mL of a diluted, digested tissue sample. Following filtration,the eluate may be propelled, e.g., by vacuum, suction, or gravity, intoa second sterile container (e.g., a wash/centrifugation bag) via a fluidpassageway preferably regulated by line clamps (e.g., butterfly lineclamps).

Isolating cells from diluted, minced and/or enzymatically digestedtissue can be accomplished by various mechanisms. In an embodiment, thetarget cells are isolated from the diluted, minced and/or enzymaticallydigested tissue by sedimentation. Although gravity sedimentation can beused, the sedimentation process can be accelerated by, for example,centrifugation. The present invention can include customized centrifugebuckets, inserts, and balance weights to ensure proper centrifuge of thesystem.

Sedimentation separates the target cells from the diluted, minced and/orenzymatically digested tissue sample. To facilitate cell collection,supernatants substantially free of cells are optionally removed via anoutlet port and a fluid passageway preferably regulated by line clamps.The supernatant may be removed by, for example, decanting or aspiration.In an example in which the second sterile container is a compressiblebag, the supernatant may be decanted by physically pressing the bag.Alternatively, the supernatant may be removed, e.g., by vacuum, suction,or gravity. Optionally, the supernatant can be removed into a wastecontainer that is connected to the second sterile container through anoutlet port and a fluid passageway regulated by line clamps. In anembodiment, the removed supernatant may be stored and/or used for otherpurposes such as maintaining cells (in culture).

To collect target cells, a small volume of a diluting solution (e.g., 20ml of autologous plasma) can be added to resuspend the cell pellet whichmay collect at the bottom of the second sterile container. As shown inthe embodiment depicted in FIG. 10A, the second sterile container canhave a bottom that is tapered to an angle sufficient to facilitatemovement of the target cells into a fluid passageway located at thebottom of the container, and optionally into a transfer container (e.g.,transfer bag). Alternatively, as depicted in FIG. 10B, target cells maybe moved into a fluid passageway located at the side of the container,and optionally into a transfer container. Movement of the cells out ofthe second sterile container and into the fluid passageway andoptionally into a transfer container can be facilitated by vacuum orsuction and may be regulated by line clamps.

If needed, the purified, target cells can be used immediately.Typically, however, the cells are cryopreserved for later use. Toachieve long-term storage, cells can be transferred from the optionaltransfer container into a sealable, sterile container amenable tofreezing (e.g., cryo-bag). Alternatively, cells can be directlycollected from the second sterile container into a freezable containerfor later use. Cryoprotectants are added to assist in storage andpreservation of target cells, and can include, for example, dimethylsulfoxide (DMSO), albumin, and/or dextran, separately or in combination.Cryoprotectants may be added to the cells within the second sterilecontainer following sedimentation. Alternatively, cryoprotectants may beadded to and mixed with the cells within the optional transfer containeror within the freezable container for long-term storage and later use.

In an embodiment, methods for separating minced and/or enzymaticallydigested tissue from undigested tissue may include two or morefiltration steps as depicted in FIGS. 10C-10D. For example, mincedand/or enzymatically digested tissue may be subjected to a firstfiltration step to remove coarse, undigested tissue. Because the mincedand/or enzymatically digested tissue is generally viscous, the tissuecan be washed or diluted with an appropriate sterile solution at anystage in the process. For example, after the minced and/or enzymaticallydigested tissue has been separated from the undigested tissue followingthe first filtration step, further washes can be performed to furthercleanse the tissue prior to a second filtration step. In an embodiment,the second filtration step may utilize a smaller sized filter in orderto remove contaminants such as collagen fibers from the minced and/orenzymatically digested tissue. Following the second filtration step,target cells may be collected by sedimentation and moved into anoptional transfer bag through a fluid passageway located either at thebottom of the container (FIG. 10C) or at the side of the container (FIG.10D). Alternatively, the cells may be directly collected into a sterilecryo-bag for long-term storage and later use.

Additional exemplary processes for separating minced tissue samples aredepicted in FIGS. 10E-10G. In each of these processes, a tissue sampleis placed in a mincer, such as the mincer of FIG. 7. In operation, themincer forces the tissue sample through one or more cutting surfaces anddeposits the finely minced tissue on the other side of the cuttingsurface(s). A saline bag is provided to permit the flushing of thetissue out of the mincer; typically, up to 500 mL of saline may be usedfor this purpose. When flushed from the mincer, the minced tissue mayflow into an optional digestion bag (as shown), where the minced tissuemay be enzymatically digested (as described with reference to FIGS.10A-10D) prior to further processing. The optional digestion bag is influid communication with a dilution bag. Alternatively, if themechanical mincing has obviated the need for any enzymatic digestion,the minced tissue can flow directly into a dilution bag. Themechanically minced tissue (whether or not subjected to enzymaticdigestion) may be viscous. The dilution bag can be mechanicallymanipulated to encourage the mixing of the tissue and the saline. Thedilution bag is also fitted with an optional injection port, permittingthe injection of additional saline into the dilution bag as required.

The tissue suspension is then filtered, once the viscosity has beensufficiently reduced. As shown in FIGS. 10E-G, the suspension passesfrom the dilution bag into a filter bag having at least one in-bagfilter. FIG. 10E depicts an embodiment with a single in-bag filter whichretains particles larger than about 40-70 μm. FIG. 10F depicts anembodiment with a single in-bag filter which retains particles largerthan about 150-250 μm. In FIG. 10F, the filtrate from the in-bag filterthen passes through a second, in-line filter unit which retainsparticles larger than about 40-70 μm. FIG. 10G depicts an embodiment inwhich the filter bag contains two in-bag filters in succession, eachin-bag filter having a surface area of at least 300 cm²; the firstfilter retains particles larger than about 500 μm and the second filterretains particles larger than about 100 μm. In each of FIGS. 10E-G, thefilter bag includes a port permitting the removal of the retentate fromthe first filter. This retentate can optionally be used as a tissueexplant for culturing cells.

The filtrates in FIGS. 10E-G pass into a centrifugation bag like thosedepicted in FIGS. 10A-D. Cells are separated from the suspension bysedimentation (e.g. by centrifugation) and are concentrated in thebottom portion of the bag, or in a fluid passageway connected to thebottom portion of the bag. The supernatant can be removed (e.g. bydecanting, aspiration, vacuum, suction, or by compressing the bag)through tubing optionally connected to a waste container. Thesupernatant may be used for other purposes such as maintaining cells inculture.

To collect target cells, a small volume of a diluting solution (e.g., 20ml of autologous plasma) can be added to resuspend sedimented cells. Asshown in FIGS. 10E-G, the resuspended cells can pass from thecentrifugation bag into a transfer bag, optionally after passing througha second filter bag, such as a second filter bag containing a filterhaving a surface area of at least 100 cm² and retaining particlesgreater than about 40 μm, as shown in FIG. 10G. The cells can betransferred to a cryobag and one or more cryoprotectants can be added,such as DMSO, albumin, and/or dextran, as described above for FIGS.10A-D.

The methods described herein are effective for purifying cells from avariety of solid tissues. For example, the methods described herein canseparate cells, such as stem cells, from fat tissue or afterbirthtissue, such as placenta or umbilical cord tissue or, more specifically,a tissue comprising Wharton's Jelly. The purified Wharton's Jelly stemcells can be used to treat or regenerate any of a variety of tissuessuch as bone, cartilage, fat or muscle. These cells can also facilitatehematopoietic engraftment and have the potential to regulate andsuppress immune responses in a host.

In addition to purified cells, the methods described herein also yieldadditional useful products. For example, when the cells are separatedfrom the minced and/or enzymatically digested tissue, the remaining,cell-depleted tissue is a rich, sterile solution that can be used formaintaining cells (in culture, for example). Further, any fragments ofundigested tissue remaining after a digestion process can also beuseful. For example, undigested umbilical cord tissue can be utilized asa seeding source for the expansion of mesenchymal stem cells.

The terms and expressions employed herein are used as terms andexpressions of description and not of limitation, and there is nointention, in the use of such terms and expressions, of excluding anyequivalents of the features shown and described or portions thereof. Inaddition, having described certain embodiments of the invention, it willbe apparent to those of ordinary skill in the art that other embodimentsincorporating the concepts disclosed herein may be used withoutdeparting from the spirit and scope of the invention. Accordingly, thedescribed embodiments are to be considered in all respects as onlyillustrative and not restrictive. Furthermore, the configurationsdescribed herein are intended as illustrative and in no way limiting.Similarly, although physical explanations have been provided forexplanatory purposes, there is no intent to be bound by any particulartheory or mechanism, or to limit the claims in accordance therewith.

INCORPORATION BY REFERENCE

The entire disclosures of each of the patent documents and scientificarticles cited herein are incorporated by reference in their entiretyfor all purposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

What is claimed is:
 1. A method of mincing a tissue sample positionedbetween a solid member and a cutting surface of a tissue mincing tool,the method comprising: pressing the tissue sample, via the solid member,into contact with and through the cutting surface of the tissue mincingtool, the tissue mincing tool comprising: a compartment for the tissuesample; the solid member moveable within the compartment; the cuttingsurface at one end of the compartment; and a sterile, sealed container,wherein the cutting surface separates the compartment from the sterile,sealed container, such that the tissue sample that passes through thecutting surface is deposited as a minced tissue within the sterile,sealed container.
 2. A method of digesting a tissue sample, the methodcomprising: mincing the tissue sample according to the method of claim 1and, optionally, transferring the minced tissue to a second sterile,sealed container; and injecting an enzyme, whereby the enzyme digeststhe minced tissue.
 3. The method of claim 2, wherein the enzyme is acollagenase.
 4. A method of separating cells from a tissue sample, themethod comprising: mincing the tissue sample according to the method ofclaim 1; and removing undigested tissue.
 5. The method of claim 4,further comprising diluting the tissue sample, wherein the undigestedtissue is subsequently removed by filtering.
 6. The method of claim 5,further comprising sedimenting a filtrate generated by the filtering. 7.The method of claim 6, further comprising re-suspending sedimented cellsand filtering the re-suspended sedimented cells.
 8. The method of claim1, wherein the cutting surface is dimensioned to mince the tissue sampleinto fragments having an average cross-section no greater than foursquare millimeters.
 9. The method of claim 1, wherein the cuttingsurface is dimensioned to mince the tissue sample into fragments, andthe tissue mincing tool further comprises a second cutting surface forreducing an average cross-section of the fragments.
 10. The method ofclaim 1, wherein the tissue mincing tool further comprises at least onemincer screen positioned in proximity to the cutting surface.
 11. Themethod of claim 10, wherein the at least one mincer screen is moveabletowards the tissue sample.
 12. The method of claim 1, wherein the tissuemincing tool further comprises a suction cup for stabilizing the tissuemincing tool during operation thereof.
 13. The method of claim 1,wherein the sterile, sealed container comprises at least one sealedaccess port permitting the sterile introduction of a fluid into thecontainer.
 14. The method of claim 1, wherein an interior surface of thecompartment is threaded.
 15. The method of claim 1, wherein the tissuemincing tool further comprises a gasket for sealing the compartment. 16.The method of claim 1, wherein a portion of the compartment near its endhas a substantially constant cross-section.
 17. The method of claim 16,wherein the solid member is shaped to mate with and to fill the portionof the compartment.
 18. The method of claim 1, wherein the cuttingsurface is rotatable.
 19. The method of claim 1, wherein the cuttingsurface is moveable towards the tissue sample.
 20. The method of claim1, wherein the tissue mincing tool further comprises a shaft crank formoving the cutting surface towards the tissue sample.
 21. The method ofclaim 1, wherein the tissue mincing tool comprises a series of cuttingsurfaces at the one end of the compartment.